Project Summary Relapse to cocaine abuse is associated with cellular and molecular adaptations in both neurons and astrocytes in the nucleus accumbens (NAc). Cocaine-induced adaptations observed in NAc astrocytes include changes in cellular morphology and synaptic contact, as well as disruptions in glutamate homeostasis. Disruptions in glutamate homeostasis lead to maladaptive changes in glutamatergic signaling and plasticity that are believed to drive relapse to cocaine use. Accordingly, elucidation of the mechanism(s) responsible for cocaine-induced disruptions in glutamate homeostasis and astroglial signaling is of great interest. Glutamate homeostasis is maintained in part by the astroglial glutamate transporter GLT-1. In animal models of cocaine use, short-access (ShA) cocaine self-administration and extinction training, as well as long-access (LgA) cocaine self- administration and withdrawal, both result in the downregulation of GLT-1 protein expression in the NAc. Moreover, restored expression of GLT-1 in the NAc prevents reinstatement to cocaine seeking. Although a decrease in GLT-1 protein is observed in both of these widely-used models, preliminary data show that a decrease in GLT-1 mRNA is only observed after LgA cocaine self-administration and withdrawal, suggesting a dual mechanism of GLT-1 regulation by cocaine. Furthermore, preliminary data shows that the GLT-1 gene is hypermethylated in the NAc after LgA cocaine self-administration and withdrawal, providing one molecular mechanism for the decrease in GLT-1 mRNA. The experiments outlined in this proposal are designed to expand on these exciting findings by further investigating the relationship between epigenetic regulation of GLT-1 expression, astrocyte morphology, and cocaine seeking after protracted withdrawal from LgA cocaine self- administration. In Aim 1, I will determine whether these preliminary data obtained from male rats also extend to female rats. To further characterize the epigenetic changes to GLT-1, Aim 1 will also assess histone modifications associated with the GLT-1 gene after LgA cocaine self-administration and withdrawal. Aim 2 will test the hypothesis that blocking DNA methylation will decrease cocaine seeking after prolonged withdrawal by a GLT-1-dependent mechanism. In Aim 3, I will test the hypothesis that AAV-mediated overexpression of GLT- 1 in astrocytes will block the cocaine-induced decrease in astrocyte surface area, volume, and synaptic localization. This will be accomplished by overexpression of GLT-1 together with a membrane-tagged GFP selectively in astrocytes so that morphometric properties of astrocytes can be measured in cells positive and negative for GLT-1 overexpression. I hypothesize that withdrawal from LgA self-administration will lead to a significant reduction in astrocyte surface area, volume, and synaptic colocalization, and that this effect will be blocked by GLT-1 overexpression. These experiments will allow for analysis of the mechanistic relationships between the effects of LgA cocaine on behavior, GLT-1 expression, and astrocyte morphology.